Dependence of p35 nuclear export on its NES.

<p>(A) Consensus sequence of the NES on p35. The functional NES comprises a core of closely spaced leucine residues or other hydrophobic amino acids. The critical hydrophobic residues in the putative NES of p35 are underlined. An NES mutant of p35, p35NES, was generated by mutating the 3 conserved hydrophobic residues (i.e., leucine 226, 227, and 230) to alanine. (B) The NES of p35 is required to mediate the nuclear export of NIF-1. COS-7 cells were co-transfected with HA-NIF-1 and p35WT or p35NES and subsequently stained with HA antibody. The results of the quantitative analysis represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C) Mutating the NES of p35 increased the population of Neuro-2A cells containing nuclear p35. Neuro-2A cells were transfected with p35WT-GFP or p35NES-GFP and subsequently differentiated by RA. The cells were stained with p35 antibody. Representative fluorescent images depicting the localization of p35WT and p35NES (indicated by the p35 staining and GFP expression). (D) Quantitative analysis of cells with nuclear p35. Cells with nuclear p35 were counted if the GFP signal in the nucleus was>75% than that of the cytoplasm. Results represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, Student's <i>t</i>-test). (E) LMB treatment caused the accumulation of p35 in the nucleus of cortical neurons. Cultured cortical neurons were treated with LMB for 1 h, and subcellular fractionation was performed. Western blot analysis of p35 and Cdk5. Total: protein extracted from the same batch of neurons using RIPA.